Breast Cancer Cell Lines BT474 Research Articles

A multiparametric screen uncovers FDA-approved small molecules that potentiate the nuclear mechano-dysfunctions in ATR-defective cells

Targeting nuclear mechanics is emerging as a promising therapeutic strategy for sensitizing cancer cells to immunotherapy. Inhibition of the mechano-sensory kinase ATR leads to mechanical vulnerability of cancer cells, causing nuclear envelope softness and collapse and activation of the cGAS-STING-mediated innate immune response. Finding novel compounds that interfere with the non-canonical role of ATR in controlling nuclear mechanics presents an intriguing therapeutic opportunity. We carried out a multiparametric high-content screen to identify small molecules that affect nuclear envelope shape and to uncover novel players that could either ameliorate or further compromise the nuclear mechanical abnormalities of ATR-defective cells. The screen was performed in HeLa cells genetically depleted for ATR. Candidate hits were also tested in combination with the chemical inhibition of ATR by AZD6738, and their efficacy was further validated in the triple-negative breast cancer cell lines BT549 and HCC1937. We show that those compounds enhancing the abnormal nuclear shape of ATR-defective cells also synergize with AZD6738 to boost the expression of interferon-stimulated genes, highlighting the power of multiparametric screens to identify novel combined therapeutic interventions targeting nuclear mechanics for cancer immunotherapy.

MiRNA-105–5p regulates the histone deacetylase HDAC2 through FOXG1 to affect the malignant biological behavior of triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC) is a specific subtype of breast cancer (BC). Some potential molecular targets have been identified, and miR-105–5p was found to be abnormally expressed in TNBC tissues. ObjectiveThe objective of this study was to probe the effect of miR-105–5p on TNBC via FOXG1/HDAC2-mediated acetylation. MethodsAn animal model of TNBC was established by injecting BC cells into the axillary area of nude mice. The levels of miR-105–5p, FOXG1, HDAC2, Bcl-2, Bax, and Ki67 were detected via RT‒qPCR, Western blotting and immunohistochemistry. Flow cytometry, CCK-8, Transwell and colony formation assays were used to measure apoptosis, proliferation and migration, respectively. Total histone acetylation levels were measured by ELISA. The binding of FOXG1 to HDAC2 was detected by co-immunoprecipitation. The binding relationship between miR-105–5p and FOXG1 was verified using a dual-luciferase reporter gene assay. ResultsIn this study, miR-105–5p and HDAC2 were highly expressed in the MDA-MB-231 and BT-549 BC cell lines, whereas FOXG1 was expressed at low levels. The inhibition of miR-105–5p inhibited the proliferation and migration of MDA-MB-231 and BT-549 cells and promoted their apoptosis. Bioinformatics analysis revealed that miR-105–5p and FOXG1 had a negative targeting regulatory relationship. FOXG1 overexpression had a similar effect on cancer cells as the inhibition of miR-105–5p. Moreover, experiments revealed that FOXG1 and HDAC2 could bind to each other and that HDAC2 overexpression or treatment with the histone acetyltransferase inhibitor Garcinol weakened the effect of FOXG1 overexpression. In addition, FOXG1 knockdown inhibited the effect of the miR-105–5p inhibitor, while Garcinol treatment further enhanced the effect of FOXG1 knockdown, inhibited histone acetylation, promoted the proliferation and migration of cancer cells, and inhibited apoptosis. Moreover, the in vivo results confirmed the in vitro results. ConclusionmiR-105–5p promotes HDAC2 expression by reducing FOXG1, inhibits histone acetylation, and aggravates the malignant biological behavior of TNBC cells.

Differential effects of docosahexaenoic acid (DHA) and linoleic acid (LA) on miR-101 and miR-342 tumor suppressor microRNAs in Taxol-treated HER2-positive breast cancer cells

Background & AimsDocosahexaenoic acid (DHA) and linoleic acid (LA) have been shown to exhibit anti-proliferative effects against breast cancer cells. However, the mechanisms underlying these effects are not yet fully understood. One potential mechanism is through the regulation of microRNAs (miRs), which are known to play a crucial role in breast cancer development and progression. This study aimed to investigate the expression of miR-342 and miR-101 as tumor-suppressor miRs in the human HER-2 positive breast cancer cell line BT-474 after treatment with DHA, LA, alone or in combination with Taxol, a standard chemotherapy agent. MethodsThe human breast cancer cell line BT-474 was cultured, and the IC50 for Taxol was determined using the MTT assay. Cells were then cultured and treated for 24 hours with 100 μM DHA and 50 μM LA, alone or in combination with the respective IC50 of Taxol. Cells were harvested, and miRNA extraction and cDNA synthesis were performed using standard methods. Expression levels of miRs were analyzed using quantitative real-time PCR (qRT-PCR), and results were normalized against U6 snRNA expression levels. ResultsThe Taxol IC50 for BT-474 cells was 19 nM. According to the data obtained from our study, it was observed that Taxol treatment resulted in the down-regulation of both miR-101 and miR-342 (3.69 (p < 0.0001) and 1.88 fold, (p < 0.0001) respectively). In addition, DHA, LA and DHA+LA caused up-regulation of miR-101 (0.11, 0.05, 0.03 fold (p < 0.0001) respectively) but not miR-342 (decreased by 1.93 (p < 0.0001), 2.89 (p < 0.0001) and 1.19 fold (p = 0.0029) respectively). Notably, treatment with DHA, LA and DHA+LA was able to restore the down-regulated expression of miR-101 (0.25 (p < 0.0001), 0.05 (p = 0.0012) and 0.06 fold (p < 0.0001) respectively) during Taxol treatment. ConclusionOur study demonstrates that DHA and LA can effectively compensate for the reduced expression of miR-101 during Taxol treatment. These findings suggest that dietary fatty acids may play a critical role in modulating the anti-cancer effects of chemotherapy agents. Future studies are needed to investigate the functional aspects of dietary fatty acids on breast cancer development and progression.

Proximity-Guaranteed DNA Machine for Accurate Identification of Breast Cancer Extracellular Vesicles.

Breast cancer is one of the most diagnosed cancers worldwide. Precise diagnosis and subtyping have important significance for targeted therapy and prognosis prediction of breast cancer. Herein, we design a proximity-guaranteed DNA machine for accurate identification of breast cancer extracellular vesicles (EVs), which is beneficial to explore the subtype features of breast cancer. In our design, two proximity probes are located close on the same EV through specific recognition of coexisting surface biomarkers, thus being ligated with the help of click chemistry. Then, the ligated product initiates the operation of a DNA machine involving catalytic hairpin assembly and clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas12a-mediated trans-cleavage, which finally generates a significant response that enables the identification of EVs expressing both biomarkers. Principle-of-proof studies are performed using EVs derived from the breast cancer cell line BT474 as the models, confirming the high sensitivity and specificity of the DNA machine. When further applied to clinical samples, the DNA machine is shown to be capable of not only distinguishing breast cancer patients with special subtypes but also realizing the tumor staging regarding the disease progression. Therefore, our work may provide new insights into the subtype-based diagnosis of breast cancer as well as identification of more potential therapeutic targets in the future.

Abstract 3071: Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors

Abstract Advances in research have shown that cancer cells have distinctive bioenergetic alterations to meet their energy requirements. It has been established that certain cancers are significantly reliant on mitochondrial respiration, suggesting that modulation of mitochondrial function may have therapeutic value. The Wnt signaling pathway is a potential therapeutic target of cancer, including breast and colorectal cancers. Recent findings suggest that bi-directional crosstalk between Wnt signaling and mitochondrial function plays a crucial role in tumorigenesis, as well as differentiation and self-renewal of stem cells. In this study, 71 agents known to alter Wnt signaling were screened for effects on mitochondrial function in BT474 and Hs578T breast cancer cell lines, which have highly oxidative and highly glycolytic bioenergetic phenotypes, respectively. This screen was performed using the Agilent Seahorse XF Mito Tox assay, which enables sensitive detection and characterization of mitochondrial modulators via a standardized, quantitative parameter, the Mito Tox Index (MTI). The MTI facilitates the comparison of drug efficacy by detecting modes of mitochondrial toxicity, inhibition, and uncoupling. The MTI-based screening of the Wnt modulators identified one inhibitor and two uncouplers as potent agents inducing mitochondrial dysfunction for both BT474 and Hs578T. Despite their distinctive bioenergetic phenotypes, MTI-based dose-response assays revealed no significant differences in the efficacy of these modulators between BT474 and Hs578T cell types. In contrast, significant differential effects on ATP production rates were detected, showing cellular energy depletion induced by these agents. Using the Agilent Seahorse XF Real-Time ATP Rate assay, BT474 cells showed significant metabolic energy crisis in the presence of all 3 compounds, whereas Hs578T cells maintained stable total ATP production. This differential impact on ATP production rate corresponds directly to differences in cell viability, with BT474 showing higher susceptibility to these compounds. These results suggest that the anti-cancer efficacy of mitochondrial modulators can be different depending on the metabolic phenotype of the target cell and should be considered in therapeutic design. Citation Format: Yoonseok Kam, Lisa Winer, George W. Rogers, Natalia Romero. Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3071.

Unveiling the Molecular Mechanism of Trastuzumab Resistance in SKBR3 and BT474 Cell Lines for HER2 Positive Breast Cancer.

HER2-positive breast cancer is one of the most prevalent forms of cancer among women worldwide. Generally, the molecular characteristics of this breast cancer include activation of human epidermal growth factor receptor-2 (HER2) and hormone receptor activation. HER2-positive is associated with a higher death rate, which led to the development of a monoclonal antibody called trastuzumab, specifically targeting HER2. The success rate of HER2-positive breast cancer treatment has been increased; however, drug resistance remains a challenge. This fact motivated us to explore the underlying molecular mechanisms of trastuzumab resistance. For this purpose, a two-fold approach was taken by considering well-known breast cancer cell lines SKBR3 and BT474. In the first fold, trastuzumab treatment doses were optimized separately for both cell lines. This was done based on the proliferation rate of cells in response to a wide variety of medication dosages. Thereafter, each cell line was cultivated with a steady dosage of herceptin for several months. During this period, six time points were selected for further in vitro analysis, ranging from the untreated cell line at the beginning to a fully resistant cell line at the end of the experiment. In the second fold, nucleic acids were extracted for further high throughput-based microarray experiments of gene and microRNA expression. Such expression data were further analyzed in order to infer the molecular mechanisms involved in the underlying development of trastuzumab resistance. In the list of differentially expressed genes and miRNAs, multiple genes (e.g., BIRC5, E2F1, TFRC, and USP1) and miRNAs (e.g., hsa miR 574 3p, hsa miR 4530, and hsa miR 197 3p) responsible for trastuzumab resistance were found. Downstream analysis showed that TFRC, E2F1, and USP1 were also targeted by hsa-miR-8485. Moreover, it indicated that miR-4701-5p was highly expressed as compared to TFRC in the SKBR3 cell line. These results unveil key genes and miRNAs as molecular regulators for trastuzumab resistance.

A peptide derived from interleukin-10 exhibits potential anticancer activity and can facilitate cell targeting of gold nanoparticles loaded with anticancer therapeutics

Human interleukin-10 (IL-10) is an immunosuppressive and anti-inflammatory cytokine, and its expression is upregulated in tumor tissues and serum samples of patients with various cancers. Because of its immunosuppressive nature, IL-10 has also been suggested to be a factor leading to tumor cells’ evasion of immune surveillance and clearance by the host immune system. In this study, we refined a peptide with 20 amino acids, named NK20a, derived from the binding region of IL-10 on the basis of in silico analysis of the complex structure of IL-10 with IL-10Ra, the ligand binding subunit of the IL-10 receptor. The binding ability of the peptide was confirmed through in vitro biophysical biolayer interferometry and cellular experiments. The IL-10 inhibitory peptide exerted anticancer effects on lymphoma B cells and could abolish the suppression effect of IL-10 on macrophages. NK20a was also conjugated with gold nanoparticles to target the chemotherapeutic 5-fluorouracil (5-FU)-loaded nanoparticles to enhance the anticancer efficacy of 5-FU against the breast cancer cell line BT-474. Our study demonstrated that NK20a designed in silico with improved binding affinity to the IL-10 receptor can be used as a tool in developing anticancer strategies.

In Vitro Evaluation of Cytotoxic and Antitumor Activities of The Tamoxifen and Doxorubicin Combination on MCF-7 and BT-474 Breast Cancer Cell Lines

The combination therapy of breast cancer has preferred for the patients to minimize possible side effects, drug resistance, recurrence and toxic effects. In this study, we aim to investigate the cytotoxic and antitumor activities the tamoxifen and doxorubicin combination in breast cancer cell lines, MCF-7 and BT-474. Tamoxifen (Tam) and doxorubicin (Dox) and their combination with different concentrations (0.625–20 μM Tam; 0.0625–2 μM Dox and 5 μM Tam+ 0.5/1.0/1.5 μM Dox combination were applied to MCF-7 and BT-474 cells for 48 hours. Afterthat, their cytotoxic activities were analyzed with MTT assay. Bcl-2, Mapt and Mrp1 are genes that induce cell proliferation, inhibit apoptosis and play role in drug resistance in cancer cells. To evaluate the antitumor activities of these genes in combination treatment, mRNA levels were analyzed by quantitative PCR. According to the MTT assay, it was determined that IC50 values as 17.26 μM and 16.65 μM for tamoxifen on MCF-7 and BT-474 breast cancer cell lines. IC50 values of doxorubicin in MCF-7 and BT-474 cells were 1.65 μM and 1.57 μM, respectively. It was found that the application of the combination drugs (15 μM tamoxifen and 1.5 μM doxorubicin) in MCF-7 and BT-474 cells have the lowest combination index values as 1.09 and 1.26, respectively. Therefore, the combination of 15 μM tamoxifen and 1.5 μM doxorubicin was selected and applied to both breast cancer cell lines for gene expression analysis. It was found that while Mrp1 and Mapt genes expressions were significantly upregulated, Bcl-2 gene expression was downregulated in MCF-7 cells. However, Bcl-2, Mrp1 and Mapt genes expressions in BT-474 cells were not significantly regulated. Altogether, these findings suggest that the combination of these two drugs may have a potential antagonistic interaction according combination index values.

MAF1 is a predictive biomarker in HER2 positive breast cancer.

RNA polymerase III transcription is pivotal in regulating cellular growth and frequently deregulated in various cancers. MAF1 negatively regulates RNA polymerase III transcription. Currently, it is unclear if MAF1 is universally deregulated in human cancers. Recently, MAF1 expression has been demonstrated to be altered in colorectal and liver carcinomas and Luminal B breast cancers. In this study, we analyzed clinical breast cancer datasets to determine if MAF1 alterations correlate with clinical outcomes in HER2-positive breast cancer. Using various bioinformatics tools, we screened breast cancer datasets for alterations in MAF1 expression. We report that MAF1 is amplified in 39% of all breast cancer sub-types, and the observed amplification co-occurs with MYC. MAF1 amplification correlated with increased methylation of the MAF1 promoter and MAF1 protein expression is significantly decreased in luminal, HER2-positive, and TNBC breast cancer subtypes. MAF1 protein expression is also significantly reduced in stage 2 and 3 breast cancer compared to normal and significantly decreased in all breast cancer patients, regardless of race and age. In SKBR3 and BT474 breast cancer cell lines treated with anti-HER2 therapies, MAF1 mRNA expression is significantly increased. In HER2-positive breast cancer patients, MAF1 expression significantly increases and correlates with five years of relapse-free survival in response to trastuzumab treatment, suggesting MAF1 is a predictive biomarker in breast cancer. These data suggest a role for MAF1 alterations in HER2-positive breast cancer. More extensive studies are warranted to determine if MAF1 serves as a predictive and prognostic biomarker in breast cancer.

In Vitro microRNA Expression Profile Alterations under CDK4/6 Therapy in Breast Cancer.

Breast cancer is the most common type of cancer worldwide. Cyclin-dependent kinase inhibition is one of the backbones of metastatic breast cancer therapy. However, there are a significant number of therapy failures. This study evaluates the biomarker potential of microRNAs for the prediction of a therapy response under cyclin-dependent kinase inhibition. This study comprises the analysis of intracellular and extracellular microRNA-expression-level alterations of 56 microRNAs under palbociclib mono as well as combination therapy with letrozole. Breast cancer cell lines BT-474, MCF-7 and HS-578T were analyzed using qPCR. A palbociclib-induced microRNA signature could be detected intracellularly as well as extracellularly. Intracellular miR-10a, miR-15b, miR-21, miR-23a and miR-23c were constantly regulated in all three cell lines, whereas let-7b, let-7d, miR-15a, miR-17, miR-18a, miR-20a, miR-191 and miR301a_3p were regulated only in hormone-receptor-positive cells. Extracellular miR-100, miR-10b and miR-182 were constantly regulated across all cell lines, whereas miR-17 was regulated only in hormone-receptor-positive cells. Because they are secreted and significantly upregulated in the microenvironment of tumor cells, miRs-100, -10b and -182 are promising circulating biomarkers that can be used to predict or detect therapy responses under CDK inhibition. MiR-10a, miR-15b, miR-21, miR-23a and miR-23c are potential tissue-based biomarkers.

Conjugation of Triterpenic Acids of Ursane and Oleanane Types with Mitochondria-Targeting Cation F16 Synergistically Enhanced Their Cytotoxicity against Tumor Cells.

The present work shows the cytotoxic effects of novel conjugates of ursolic, oleanolic, maslinic, and corosolic acids with the penetrating cation F16 on cancer cells (lung adenocarcinoma A549 and H1299, breast cancer cell lines MCF-7 and BT474) and non-tumor human fibroblasts. It has been established that the conjugates have a significantly enhanced toxicity against tumor-derived cells compared to native acids and also demonstrate selectivity to some cancer cells. The toxic effect of the conjugates is shown to be due to ROS hyperproduction in cells, induced by the effect on mitochondria. The conjugates caused dysfunction of isolated rat liver mitochondria and, in particular, a decrease in the efficiency of oxidative phosphorylation, a decrease in the membrane potential, and also an overproduction of ROS by organelles. The paper discusses how the membranotropic- and mitochondria-targeted effects of the conjugates may be related to their toxic effects.

WNT-Conditioned Mechanism of Exit from Postchemotherapy Shock of Differentiated Tumour Cells

Background: the present study aims to prove or disprove the hypothesis that the state of copy number aberration (CNA) activation of WNT signalling pathway genes accounts for the ability of differentiated tumour cells to emerge from postchemotherapy shock. Methods: In the first step, the CNA genetic landscape of breast cancer cell lines BT-474, BT-549, MDA-MB-231, MDA-MD-468, MCF7, SK-BR-3 and T47D, which were obtained from ATCC, was examined to rank cell cultures according to the degree of ectopic activation of the WNT signalling pathway. Then two lines of T47D with ectopic activation and BT-474 without activation were selected. The differentiated EpCAM+CD44-CD24-/+ cells of these lines were subjected to IL6 de-differentiation with formation of mammospheres on the background of cisplatin and WNT signalling inhibitor ICG-001. Results: it was found that T47D cells with ectopic WNT signalling activation after cisplatin exposure were dedifferentiated to form mammospheres while BT-474 cells without ectopic WNT-signalling activation did not form mammospheres. The dedifferentiation of T47D cells after cisplatin exposure was completely suppressed by the WNT signalling inhibitor ICG-001. Separately, ICG-001 reduced, but did not abolish, the ability to dedifferentiate in both cell lines. Conclusions: these data support the hypothesis that the emergence of differentiated tumour cells from postchemotherapy shock after chemotherapy is due to ectopic activation of WNT signalling pathway genes.

Cultivation of cells in alginate drops as a high-performance method of obtaining cell spheroids for bioprinting

Purpose of the study. Testing the protocol of obtaining cell spheroids of breast cancer cell cultures for bioprinting by growing in alginate drops.Materials and methods. Cells of breast cancer cell lines BT-20 and MDA-MB-453 were cultured in DMEM medium supplemented with 10 % FBS. Next, the cells were removed from the plastic using a trypsin-V ersene solution and resuspended in a sterile 2 % alginate solution in DPBS to the concentration of 105 cells/ml. Then the alginate solution with the cells was slowly dripped through a 30G needle into a sterile cooled solution of calcium chloride (100 mM) from a height of 10 cm. After polymerization, alginate drops were washed in DMEM and cultured for two weeks in DMEM with the addition of 10 % FBS at 37 °C and 5.0 % CO2.The spheroids formed in the alginate were photographed on the 3rd, 7th, 10th, and 14th days of cultivation, after which they were removed from the alginate by keeping in 55 mM sodium citrate solution with the addition of 20mM ethylenediaminetetraacetic acid (EDTA) and embedded in paraffin blocks according to the standard method, followed by histological examination.Results. Cellular spheroids were formed in both cell cultures already on the 3rd day of cultivation. From the 3rd to the 10th day in both cultures, a uniform growth of cell spheroids was observed with a gradual slowdown in the increase in the size of spheroids by the 14th day of cultivation. On the 10th day the proportion of cells that formed clones (more than 500 μm2 in size) was 25.2 % ± 7.1 % (n = 25) in the BT-20 culture and 38.5 % ± 9.9 % (n = 25) in MDA-MB-453 culture. On the 14th day, BT-20 culture was characterized by spheroids varying little in size and shape, with an average area of 1652 ± 175 µm2, having a dense structure with smooth edges. The spheroids in MDA-MB-453 culture turned out to be more loose and easily deformed, their size and shape varied noticeably, the average area of the spheroids was 2785 ± 345 µm2.Conclusion. The production of spheroids in alginate drops is inferior in speed to the methods of forming cell conglomerates in hanging drops or on microwells, but it surpasses these methods in productivity, which is comparable to the production of spheroids by constant medium stirring on low-adhesive substrates. In addition, the clonal nature of the obtained spheroids leads to an increase in research costs and thus limits their scalability.

FOXD1 is associated with poor outcome and maintains tumor-promoting enhancer-gene programs in basal-like breast cancer.

Breast cancer biology varies markedly among patients. Basal-like breast cancer is one of the most challenging subtypes to treat because it lacks effective therapeutic targets. Despite numerous studies on potential targetable molecules in this subtype, few targets have shown promise. However, the present study revealed that FOXD1, a transcription factor that functions in both normal development and malignancy, is associated with poor prognosis in basal-like breast cancer. We analyzed publicly available RNA sequencing data and conducted FOXD1-knockdown experiments, finding that FOXD1 maintains gene expression programs that contribute to tumor progression. We first conducted survival analysis of patients grouped via a Gaussian mixture model based on gene expression in basal-like tumors, finding that FOXD1 is a prognostic factor specific to this subtype. Then, our RNA sequencing and chromatin immunoprecipitation sequencing experiments using the basal-like breast cancer cell lines BT549 and Hs578T with FOXD1 knockdown revealed that FOXD1 regulates enhancer-gene programs related to tumor progression. These findings suggest that FOXD1 plays an important role in basal-like breast cancer progression and may represent a promising therapeutic target.

Abstract P1-13-19: ESAM reduces anti-HER2 therapy sensitivity by activating mTOR pathway in HER2 positive breast cancer

Abstract Background: Trastuzumab combined with pertuzumab is the main therapy for HER2 positive breast cancer, but poor patient response due to drug resistance remains a clinical challenge. Organoids derived from tumor patients can highly maintain the heterogeneity of the original tumor, and have been used in drug sensitivity testing and new drug development. To screen for genes that associated with trastuzumab or pertuzumab drug resistance, this study used HER2-positive breast cancer organoids to establish a model of trastuzumab combined with pertuzumab and conducted high-throughput sequencing to screen the differential genes. We carried out further research to explore the effect of ESAM on drug sensitivity of HER2-targeted therapy and clarify the specific regulatory mechanisms and potential therapeutic targets of ESAM. Method: 8 HER2 positive breast cancer organoids were cultured. Immunohistochemistry, hematoxylin-eosin staining and Immunofluorescence were performed to identify the consistency between organoids and primitive tumors. According to drug sensitivity results of organoids to trastuzumab combined with pertuzumab, organoids were divided into a relatively sensitive group and a relatively insensitive group. Trastuzumab combined with pertuzumab was regularly used to stimulate the two groups. After 3 cycles, high-throughput transcriptome sequencing was used to evaluate their gene expression to find a gene responsible for trastuzumab and pertuzumab resistance. METABRIC and our center database were used to determine the prognostic value of ESAM. ESAM overexpression and knockdown stably transfected cell lines were constructed in SK-BR-3 and BT474 breast cancer cell lines by lentivirus. Colony formation assay, CCK-8 assay, organoid model and mouse xenograft model were conducted to examine the influence of ESAM on proliferation and trastuzumab or pertuzumab sensitization in vitro and in vivo. RNA-seq and GSEA analysis were performed to investigate the downstream pathways of ESAM. Western blot was used to confirm the relationship between ESAM and mTOR pathway. Small interfering RNA and mTOR/PI3K pathway inhibitors were used to confirm the function of mTOR in ESAM-induced drug resistance of trastuzumab. Results: HER2 positive breast cancer organoids can maintain the pathological characteristics of primitive tumors. The results of high-throughput transcriptome show that ESAM is significantly up-regulated in the relatively insensitive group compared to the relatively sensitive group. METABRIC and our center database suggest that high ESAM mRNA expression was associated with poor OS, RFS, DFS and PFS of HER2-positive breast cancer patients. CCK-8 and cell colony formation assay confirm that ESAM can promote the proliferation of HER2-positive breast cancer cells and inhibit the drug sensitivity of HER2-positive breast cancer cells to trastuzumab and pertuzumab. GSEA analysis shows high ESAM can activate mTORC1 signaling pathway. Western blotting analysis proves the expression of ESAM is positively correlated with mTOR pathway. Inhibition of mTOR pathway in ESAM overexpression cells can suppress ESAM-mediated proliferation and reverse the drug sensitivity of cells to trastuzumab and pertuzumab. In vivo, overexpression of ESAM can promote the proliferation of mouse mammary tumors and reduce the sensitivity of mouse mammary tumors to trastuzumab, combined use of PI3K inhibitor could reverse drug sensitivity of mouse mammary tumors to trastuzumab. Conclusion: High ESAM expression is associated with poor prognosis in patients with HER2 positive breast cancer. ESAM can activate mTOR pathway, promote cell proliferation and reduce the sensitivity of HER2 positive breast cancer cells to trastuzumab and pertuzumab, which can be inhibited by the application of PI3K/mTOR inhibitors. Keywords: ESAM; HER2 positive breast cancer; HER2 targeted therapy; mTOR; drug sensitivity. Citation Format: Jiong Wu, Xujie Zhou, Yayun Chi, Jingyan Xue. ESAM reduces anti-HER2 therapy sensitivity by activating mTOR pathway in HER2 positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-19.

Development, validation, and evaluation of a deep learning model to screen cyclin-dependent kinase 12 inhibitors in cancers

Deep learning-based in silico alternatives have been demonstrated to be of significant importance in the acceleration of the drug discovery process and enhancement of success rates. Cyclin-dependent kinase 12 (CDK12) is a transcription-related cyclin-dependent kinase that may act as a biomarker and therapeutic target for cancers. However, currently, there is no high selective CDK12 inhibitor in clinical development and the identification of new specific CDK12 inhibitors has become increasingly challenging due to their similarity with CDK13. In this study, we developed a virtual screening workflow that combines deep learning with virtual screening tools and can be applied rapidly to millions of molecules. We designed a Transformer architecture Drug-Target Interaction (DTI) model with dual-branched self-supervised pre-trained molecular graph models and protein sequence models. Our predictive model produced satisfactory predictions for various targets, including CDK12, with several novel hits. We screened a large compound library consisting of 4.5 million drug-like molecules and recommended a list of potential CDK12 inhibitors for further experimental testing. In kinase assay, compared to the positive CDK12 inhibitor THZ531, the compounds CICAMPA-01, 02, 03 displayed more effective inhibition of CDK12, up to three times as much as THZ531. The compounds CICAMPA-03, 05, 04, 07 showed less inhibition of CDK13 compare to THZ531. In vitro, the IC50 of CICAMPA-01, 04, 05, 06, 09 was less than 3 μM in the HER2 positive CDK12 amplification breast cancer cell line BT-474. Overall, this study provides a highly efficient and end-to-end deep learning protocol, in conjunction with molecular docking, for discovering CDK12 inhibitors in cancers. Additionally, we disclose five novel CDK12 inhibitors. These results may accelerate the discovery of novel chemical-class drugs for cancer treatment.

AMPK activation attenuates HER3 upregulation and Neuregulin-Mediated rescue of cell proliferation in HER2-Overexpressing breast cancer cell lines exposed to lapatinib

Lapatinib is a highly selective reversible inhibitor of the tyrosine kinase domains of HER2 and EGFR, approved for the treatment of advanced stage HER2-overexpressing breast cancers. Although targeted therapy with lapatinib provides initial clinical advantage, cancer cells' adaptive responses can overcome the inhibitory effects of lapatinib. HER3 upregulation and autocrine induction of HER3 ligand neuregulin-1 (NRG), have been implicated in the restoration of AKT and ERK1/2 activity and rescue of cell proliferation. In this study we evaluated the effects of lapatinib alone and in combination with AMPK activator GSK-621 in HER2-overexpressing breast cancer cell lines SKBR3 and BT474. Our results show that in cells exposed to lapatinib and GSK-621 in combination, lapatinib-mediated HER3 upregulation was reduced and reactivation of AKT and ERK1/2 kinases was prevented. The two drugs in combination decreased cell viability in a synergistic manner and greatly reduced the ability of NRG to rescue cell proliferation. Finally, we provide evidence that in cells exposed to lapatinib and GSK-621 in combination the establishment of a transcriptionally permissive chromatin structure at the HER3 promoter is hampered. The results of this study highlight a potential role for AMPK activation in counteracting lapatinib-induced adaptive responses of HER2-overexpressing breast cancer cells.

The mitochondrial enzyme FAHD1 regulates complex II activity in breast cancer cells and is indispensable for basal BT-20 cells in vitro.

The mitochondrial enzyme fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) was identified to be upregulated in breast cancer tissues. Here, we show that FAHD1 is indispensable for the survival of BT-20 cells, representing the basal breast cancer cell type. A lentiviral knock-down of FAHD1 in the breast cancer cell lines MCF-7 and BT-20 results in lower succinate dehydrogenase (complex II) activity. In luminal MCF-7 cells, this leads to reduced proliferation when cultured in medium containing only glutamine as the carbon source. Of note, both cell lines show attenuated protein levels of the enzyme glutaminase (GLS) which activates programmed cell death in BT-20. These findings demonstrate that FAHD1 is crucial for the functionality of complex II in breast cancer cells and acts on glutaminolysis in the mitochondria.

Synthesis, Spectroscopic Analysis, and In Vitro Anticancer Evaluation of 2-(Phenylsulfonyl)-2H-1,2,3-triazole

The 1,2,3-Triazole derivatives containing the sulfonyl group have proved their biological importance in medicinal chemistry and drug design. In this sense, we describe the regioselective synthesis of 2-(phenylsulfonyl)-2H-1,2,3-triazole 3 in good yield through a classical sulfonamidation reaction of 1H-1,2,3-triazole 1 with benzenesulfonyl chloride 2 in dichloromethane using a slight excess of triethylamine at 20 °C for 3 h. This procedure is distinguished by its short reaction time, high yield, excellent regioselectivity, clean reaction profile, and operational simplicity. The sulfonamide 3 was characterized by high-resolution mass spectrometry, FT–IR, UV–Vis, 1D and 2D NMR spectroscopy, and elemental analysis. The sulfonamide 3 exhibited moderate activity against UO-31 renal, SNB-75 central nervous system, HCT-116 colon, and BT-549 breast cancer cell lines, with growth inhibition percentages (GI%) ranging from 10.83% to 17.64%.

Abstract 3127: Negative impact of paclitaxel on human breast tumor microenvironment and its reversal by the combination of interferon-α with TLR3 agonist rintatolimod

Abstract Background: Taxanes are extensively used in the chemotherapy of breast cancer in the neoadjuvant, adjuvant and metastatic settings. Paclitaxel is known to have both immunostimulatory and immunosuppressive effects, but its impact on the chemokine system within the tumor microenvironment (TME) remains unclear. In the breast TME treated with neoadjuvant taxane chemotherapy, high levels of the cytotoxic T cell (CTL) attractants CXCL9, CXCL10 and their common receptor CXCR3 predict higher probability of pathological complete response (pCR) and improved long term outcomes. We previously developed a chemokine modulatory regimen (CKM) combining interferon-α with TLR3 agonist rintatolimod to selectively increase CTL-attracting chemokines in the TME but reduce the production of chemokines attracting myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) in the TME. Here we evaluated paclitaxel’s impact on chemokine production in the human breast TME and CKM’s ability to eliminate undesirable aspects of taxane chemotherapy. Methods: Fresh breast cancer tissues obtained during routine surgeries, breast cancer cell lines BT-549 and MDA-MD-231 and peripheral blood monocyte-derived macrophages were analyzed immediately or cultured ex vivo for 24 hours in the absence or presence of paclitaxel and/or CKM components. The expression of chemokine genes and secretion of chemokines by freshly harvested and ex vivo-treated tumor explants, cancer cell lines and macrophages were analyzed by quantitative PCR (Taqman) and ELISA. Paired student t-test was used for statistical analyses. Results: Breast cancer explants spontaneously expressed high levels of MDSC/Treg-attractants CXCL12 and CCL22, but only marginal levels of CTL and natural killer (NK) cell attractants CXCL9, CXCL10, CXCL11 and CCL5. Unexpectedly, paclitaxel treatment resulted in further elevation of granulocyte/MDSC-attractant CXCL8 and CCL22, which was reversed by the combination of paclitaxel with CKM. At the same time, while paclitaxel alone did not induce any of the CTL attractants tested, its combination with the CKM was highly effective in inducing CXCL9, CXCL10, CXCL11 and CCL5. Conclusions: Our results identify an undesirable aspect of paclitaxel’s impact on breast cancer. The ability of CKM to enhance the expression of CTL/NK cell attractants and suppress the production of Treg/MDSC attractants produced by paclitaxel provides a strong rationale for combined use of CKM in taxane-based chemo-immunotherapy of breast cancer and potentially other diseases. Citation Format: Shipra Gandhi, Melissa Grimm, Ronald Slomba, Kazuaki Takabe, Pawel Kalinski. Negative impact of paclitaxel on human breast tumor microenvironment and its reversal by the combination of interferon-α with TLR3 agonist rintatolimod [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3127.